Amlexanox attenuates LPS-induced neuroinflammatory responses in microglial cells via inhibition of NF–κB and STAT3 signaling pathways

Amlexanox is an anti-inflammatory and anti-allergic agent used clinically for the treatment of aphthous ulcers, allergic rhinitis, and asthma. Recent studies have demonstrated that amlexanox, a selective inhibitor of IkB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1), suppresses a range of diseases or inflammatory conditions, such as obesity-related metabolic dysfunction and type 2 diabetes. However, the effects of amlexanox on neuroinflammatory responses to amlexanox have not yet been comprehensively studied. In this study, we investigated the novel therapeutic effect of amlexanox on LPS-induced neuroinflammation in vivo, and intraperitoneal injection of amlexanox markedly reduced LPS-induced IKKε levels, proinflammatory cytokines, and microglial activation, as evidenced by ionized calcium-binding adapter molecule 1 (Iba1) immunostaining. Furthermore, amlexanox significantly reduced proinflammatory cytokines and chemokines in LPS-induced bone marrow-derived macrophages (BMDM), murine BV2, and human HMC3 microglial cells. This data provided considerable evidence that amlexanox can be used as a preventive and curative therapy for neuroinflammatory and neurodegenerative diseases. In terms of mechanism aspects, our results demonstrated that the anti-inflammatory action of amlexanox in BV2 microglial cells was through the downregulation of NF-κB and STAT3 signaling pathways. In addition, the combination of amlexanox and SPI (a STAT3 selective inhibitor) showed high efficiency in inhibiting the production of neurotoxic and pro-inflammatory mediators. Overall, our data provide rational insights into the mechanisms of amlexanox as a potential therapeutic strategy for neuroinflammation-related diseases.

Neuroinflammation is a process associated with the onset and progression of several neurodegenerative disorders.This process depends on the innate immune system in the central nervous system (CNS), in which microglia are the principal and initial responders to pathological insults 1 .Normally, microglia are primary mediators in the overall protection of the CNS-they are constantly hunting the CNS for plaques, injured or unnecessary neurons and synapses, and infectious agents 2 .However, irregularly activated microglia expressively accelerate neuroinflammatory and neurotoxic responses by releasing several proinflammatory factors [2][3][4] .These persistent inflammatory responses are strongly associated with the most degenerative nerve diseases, such as Alzheimer's disease, and lead to neuronal cell death, synaptic degeneration, and cognitive dysfunction 5 .Therefore, controlling microglia-mediated inflammation has been considered a crucial strategy in neuroinflammation-related disease therapy.
Multiple signal transduction pathways are involved in neuroinflammation caused by activated microglia.Many studies have reported that lipopolysaccharide (LPS), a prominent outer membrane component of gramnegative bacteria, is well known as an exogenous inducer of microglia-mediated neuroinflammation 6,7 .LPS binding to TLR4 on the microglia surface activates several signal transduction pathways, which include mitogenactivated protein kinase (MAPK), and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), which eventually lead to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation 8,9 .Activation of NF-κB then mediates the production of inducible enzymes, pro-inflammatory cytokines, and chemokines, which all together result in neuroinflammation.In addition, several studies have shown that signal transducers and activators of transcription 3 (STAT3) mediate inflammatory responses and are associated with several neurodegenerative diseases 10 .Particularly, activation of STAT3 induces neuronal apoptosis through increased TNF-α expression in the diabetic hippocampus 11 .Hence, a deeper understanding of how the immune system processes information and senses pathogens could provide a better way to treat neurodegenerative diseases.
Both TANK-binding kinase 1 (TBK1) and its homolog, IκB kinase epsilon (IKKε), are activated by phorbol esters (PMA), LPS, and cytokines [12][13][14] .TBK1/IKKɛ activity might promote the activation of various signaling pathways and has been linked to the pathology of inflammatory diseases 15 .In the nervous system, over-expression of IKKɛ has been found in the spinal cord, as well as in neurons and glial cells after inflammatory stimulation in mice 16 .Knockout or pharmacological inhibition of IKKε attenuates pain-like behavior in the spared nerve injury model of neuropathic pain 17 .TBK1 depletion suppresses nociceptive effects in inflammation models 18 .These studies indicate that TBK1/IKKε plays a certain role in the formation or progression of neurological diseases.
In clinical trials, amlexanox, an anti-inflammatory and anti-allergic immunomodulator, was used to treat aphthous ulcers 18 .Moreover, amlexanox, a specific inhibitor of TBK1/IKKɛ, has been shown in other trials to be a prospective medicine of choice for the treatment of inflammation-related illnesses such as liver fibrosis and damage, type 2 diabetes, and obesity-related metabolic dysfunction [19][20][21][22] .However, whether amlexanox can modulate neuroinflammation has been rarely reported, and is poorly understood.In the present study, we determined the effects of amlexanox on microglial activation, which plays a critical role in neuroinflammation as well as in neurodegeneration.

Amlexanox alleviated LPS-induced neuroinflammation in vivo
To investigate the effects of amlexanox on LPS-induced neuroinflammation in vivo, wild-type (WT) mice were given amlexanox (50 mg/kg, i.p.) for 3 days before being given LPS (10 mg/kg, i.p.) or phosphate-buffered saline (PBS) (Fig. 1A).Following amlexanox treatment, the mRNA expression level of IKKε but not TBK1 was significantly reduced in LPS-induced injured brain (Fig. 1B).As shown in Fig. 1C and D, LPS-injected WT mice also had higher levels of TNF-α, IL-1β, and IL-6 than vehicle-injected WT mice.Interestingly, amlexanox treatment significantly attenuated LPS-induced cytokine production, including TNF-α, IL-1β, and IL-6.It is worth noting that the amount of IKKε expression in the brain increased significantly following LPS injection.However, the regulation of TBK1 was shown to be minimal.This finding is consistent with a recent study finding IKKε activation in the spared nerve injury (SNI) model 17 .Following treatment with amlexanox, the mRNA expression level of IKKε was markedly reduced in injured brains induced by LPS.Furthermore, activated microglia cells are also important contributors to neuroinflammation.To examine the involvement of microglia in LPS-induced neuroinflammation, the expression of the ionized calcium-binding adaptor molecule (Iba-1) was determined by immunohistochemistry.The number of Iba1-reactive cells increased significantly in LPS-injected animals.Amlexanox treatment considerably reduced the activating impact of LPS on microglia.Treatment with amlexanox significantly attenuated the stimulating effect of LPS on microglia (Fig. 1E).Overall, these findings support the hypothesis that amlexanox inhibits IKKε and attenuates LPS-induced neuroinflammation by modulating microglial activation and inflammatory mediators.

Amlexanox regulates proinflammatory enzymes in LPS-stimulated BV2 microglia and bone-marrow-derived macrophage (BMDM)
To investigate the effects of amlexanox in vitro, we initially measured its cytotoxicity towards BV2 microglial cells.For this experiment, BV2 microglial cells were treated for 24 h with increasing concentrations of amlexanox or vehicle in the presence or absence of LPS, and cell viability was assessed using CCK-8 assays.In both normal and LPS-stimulated conditions, amlexanox did not induce BV2 microglial cell toxicity up to 6 μM (Fig. 2A).Therefore, amlexanox was used at concentrations less than or equal to 6 μM in subsequent studies to avoid toxic effects on BV2 microglial cells.Similar to Fig. 1B, the mRNA expression and protein levels of IKKε were significantly reduced in LPS-activated microglia after treatment with amlexanox (Supplementary Figure S1), but the TBK1 levels were not significantly changed (Fig. 2B, C).Next, activated glial cells produce inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which are directly toxic to neurons.Thus, to examine the effects of amlexanox on LPS-elicited expression of iNOS and COX-2, BV2 microglial cells were pre-treated with amlexanox for 1 h, and then treated with LPS.After 6 h, qRT-PCR was conducted to measure the mRNA expression levels of iNOS and COX-2.After 23 h, western blot was conducted to measure the protein expression levels of iNOS and COX-2.Figure 2D and F show that LPS dramatically induced iNOS and COX-2 expression levels.Treatment with amlexanox markedly suppressed LPS-induced iNOS and COX-2 mRNA expression levels in both microglia and BMDM.At the translation level, amlexanox significantly inhibited iNOS protein expression levels in a dose-dependent manner, but not COX-2 in microglia cells (Fig. 2E and supplementary Figure S2).These data suggest that amlexanox effectively attenuated iNOS expression but had an extremely limited effect on COX-2.

Amlexanox suppresses proinflammatory mediators in LPS-stimulated murine BV2, BMDM, and HMC3 human microglial cells
To investigate the potential regulatory effects of amlexanox on proinflammatory factors, BV2 microglial cells were treated with vehicle or amlexanox for 1 h, followed by LPS or PBS treatment for 6 h.Total RNA was isolated,

Amlexanox significantly inhibits NF-κB activity in LPS-stimulated BV2 microglial cells
To further investigate the underlying molecular mechanisms of the anti-inflammatory effects of amlexanox, we examined the levels of NF-κB, the main transcription factor modulating proinflammatory gene expression in nuclei.Western blotting analysis was performed to measure NF-κB p65 and its regulator IκBα activity.Our data indicated that LPS treatment strongly stimulated the phosphorylation of IκBα and p65 (Supplementary Figure S4).Amlexanox significantly suppressed the phosphorylation of both IκBα and NF-κB p65 during LPSstimulated microglia (Fig. 5A).To further confirm these findings, immunostaining was performed with anti-NF-κB p65.We observed that LPS induced the nuclear translocation of p65 to the nucleus, and the translocation was prevented by amlexanox treatment (Fig. 5B).These results demonstrate that amlexanox inhibits NF-κB activity, which may further attenuate proinflammatory mediator expression in LPS-evoked microglial activation.

Attenuation of LPS-induced BV2 microglial activation by amlexanox is partly dependent on STAT3 signaling pathway
STAT3 has been reported to be a transcription factor that plays a critical role in neuroinflammatory responses 25 .
To examine the effect of amlexanox on the STAT3 signaling pathway, BV2 microglial cells were treated with amlexanox or vehicle for 1 h, followed by treatment with LPS for 23 h.Western blotting analysis was performed to measure STAT3 phosphorylation.We found that amlexanox significantly reduced LPS-induced phosphorylation of STAT3 in BV2 microglial cells in a dose-dependent manner (Fig. 6A and Supplementary Figure S5).To determine the involvement of STAT3 in the neuroinflammatory responses, SPI (a selective STAT3 inhibitor) was administered to BV2 cultures for 1 h before being treated with amlexanox and then stimulated with LPS for 22 h. Figure 6B shows the inhibitory effect of SPI on the expression of STAT3.Treatment with SPI (5 μM) significantly attenuated LPS-induced TNF-α, CCL2, and CXCL10 (Fig. 6C).In addition, the results showed that a combination of amlexanox and SPI further impaired LPS-elicited proinflammatory mediators compared with treatment with amlexanox or SPI alone.Interestingly, LPS induced the expression of IL-1β, and IL-6 was significantly decreased in the combined treatment, which did not show in a single treatment.These data indicate that amlexanox provided anti-inflammatory effects on activated microglia, whose effects are, at least partially, dependent on STAT3 inhibition.

Post-treatment with amlexanox alters LPS-stimulated proinflammatory responses in BV2 microglial cells
We finally investigated whether post-treatment with amlexanox alters LPS-stimulated proinflammatory responses.Again, treatment with amlexanox alone did not affect the levels of any proinflammatory factors compared with the vehicle-treated control.However, in the LPS-stimulated condition, post-treatment with amlexanox significantly decreased the proinflammatory mediators (Fig. 7).Thus, these results indicate that pre-treatment (as a preventive measure) as well as post-treatment (as a curative measure) with amlexanox effectively regulates LPS-stimulated proinflammatory responses in BV2 microglial cells.

Discussion
Previous studies have shown that amlexanox mitigates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and T-cell responses 24 .Inhibition of IKKε by amlexanox impairs neuropathic pain after spared nerve injury in mice 23 .Additionally, amlexanox showed high permeability to cross the blood-brain barrier 26 .These studies raised the potential of utilizing amlexanox as a novel pharmaceutical agent for neurological disorders.Nevertheless, the effects of amlexanox on microglia, which are the primary immune effector cells in the CNS, as well as those of amlexanox on LPS-induced neuroinflammation, remain largely undisclosed.In the present study, we investigated whether amlexanox can modulate LPS-induced neuroinflammatory responses in WT mice.Interestingly, amlexanox reduced LPS-evoked proinflammatory cytokine levels of TNF-α, IL-1β, and IL-6 in both serum and brain tissue.Amlexanox consequently affected the phenotypic activation of microglia, as shown by the reduced microglia Iba-1 positive cells depicted via immunohistochemistry.Other studies revealed that LPS-induced neuroinflammation in the mouse brain is evidenced by microglial activation, which is a consequence of the robust expression of proinflammatory cytokines such as IL-1β, IL-6, and TNFα [27][28][29][30][31] .These in vivo data suggest that amlexanox can be a potential drug for neuroinflammation-related diseases.Experimental evidence suggests that resident microglia are over-activated in both acute and chronic neurological disorders and produce a plethora of proinflammatory mediators, which exacerbate brain tissue damage [32][33][34] .Of these, expression of iNOS and COX-2 was found primarily within the brain glia of the substantia nigra of post-mortem Parkinson's disease patients 35 .In the present study, amlexanox treatment significantly inhibited both the transcription and translation of iNOS, but only the transcription of COX-2 in LPS-stimulated microglial cells was inhibited.These effects were not due to any cytotoxicity of amlexanox, as verified by the CCK8 assay.Once expressed, iNOS continuously produces high levels of nitric oxide that cause excitotoxic neuronal death.Blocking the activity of iNOS has been demonstrated to prevent dopamine neuron degeneration and microglial activation in a model of Parkinson's disease 36 .These results indicate that amlexanox is a potential drug for neurodegenerative diseases, such as Parkinson's disease, through the down-regulation of iNOS.
Amlexanox itself does not affect the levels of any proinflammatory mediator under basal conditions but significantly inhibits the generation of TNF-α, CCL2, and CXCL10 by suppressing mRNA expression under LPS stimulation.Indicating that the inhibitory action of amlexanox on the production of inflammatory mediators occurs at the transcriptional level (Fig. 3).Unlike iNOS, TNF-α utilises indirect means to kill and damage neurons through glutamate excitotoxicity and glia activation 37 , while the over-expression of CCL2 and CXCL10 chemokines induces inflammatory cell infiltration to amplify inflammatory responses and increase neuronal toxicity 38 .Accordingly, the modulation of inflammatory mediators by amlexanox is important, not only for the attenuation of microglial activation but also for inflammation-mediated neuronal damage and the infiltration of blood inflammatory cells.Although IL-1β and IL-6 are typical pro-inflammatory cytokines that contribute to glial activation, amlexanox at 6 μM (the highest concentration) had a very limited effect on their mRNA   shown that 12.5 μM of amlexanox significantly inhibits the expression of IL-1β in LPS-stimulated Kupffer cells (resident liver macrophages) 22 .These findings suggest that amlexanox regulates LPS-induced proinflammatory responses in microglia, and that its effect may depend on the target cell types and species.We also showed that post-treatment with amlexanox markedly reduced a range of proinflammatory mediators, similar to pretreatment (Fig. 7).This result both confirmed the anti-inflammatory action of amlexanox in microglia and highlighted the potential of using amlexanox as preventive, as well as curative, therapy for neuroinflammatory and neurodegenerative diseases.Previous studies indicated that IKKε is expressed in the sciatic nerve, dorsal root ganglia, and spinal cord and that it is upregulated during the progression of sciatic nerve injury neuropathy 17 .In our experimental model, elevated expression of IKKε in LPS-induced neuroinflammation in the brain of mice and BV2 microglial cells was observed.Our findings suggest that IKKε contributes to the development of neuroinflammation, potentially by initiating microglial activation.Inhibition of IKKε by amlexanox resulted in the downregulation of proinflammatory mediators and neurotoxicity factors both in vitro and in vivo models (Figs. 1, 2, and 3).To further understand the anti-neuroinflammatory mechanisms of amlexanox in microglia, we began evaluating the www.nature.com/scientificreports/potential downstream signals.AKT and MAPKs activation has been reported to be regulated by TBK1/IKKε [39][40][41] .
In addition, treatment with amlexanox significantly attenuates the phosphorylation of AKT and MAPKs in experimental autoimmune encephalomyelitis and spared nerve injury disease models 23,24 .In accordance with these findings, we showed that amlexanox significantly diminished LPS-induced phosphorylation of AKT and p38 MAPK in BV2 microglia, but not JNK or ERK MAPK (Fig. 4).In addition, AKT and/or p38 MAPK have been implicated in a signal transduction pathway responsible for the activation of NF-κB [42][43][44] .We, therefore, screened the NF-κB: a ubiquitous transcription factor that has long been considered a prototypical proinflammatory signaling pathway 45 .As expected, amlexanox impaired LPS-induced activation of the NF-κB signaling pathway, as manifested by inhibition of the phosphorylation of both IκBα and p65 and NF-κB nuclei translocation (Fig. 5).Thus, inhibition of TBK1/IKKε by amlexanox might attenuate NF-κB activity through the downregulation of AKT and p38 MAPK.On the other hand, IKKε could also directly phosphorylate NF-κB p65 serine 536 46 .In addition, several studies have reported that TBK1/IKKε inhibitor inactivates NF-κB p65 complex, without mentioning their intermediate protein 22,47 .Hence, it is also possible that the inhibition of TBK1/IKKε by amlexanox directly attenuates NF-κB activity.Indeed, the regulation of NF-κB by TBK1/IKKε has long been a controversial topic 16,19 .Therefore, more detailed studies are needed to elucidate the crosstalk between TBK1/IKKε and NF-κB.Abnormal activation of STAT3 signaling was recently found to be associated with neurodegenerative disorders such as Huntington's disease or Alzheimer's disease 12 .The maintenance of a homeostatic balance in STAT3 signaling plays an important role in enabling inflammation-free conditions.In our study, LPS-induced phosphorylation of STAT3 was dramatically reduced with amlexanox treatment in BV2 microglial cells (Fig. 6A).Consistent observations were previously reported, where silencing of IKKε leads to impaired activity of STAT3 48 .In addition, amlexanox shows protective effects against fibro-inflammatory responses through the inhibition of STAT3 in the liver 22 .As such, to address the functional roles of STAT3 in neuroinflammatory responses, a STAT3-specific inhibitor (SPI) was administered to LPS-stimulated BV2 microglia.Our data showed that SPI effectively blocked STAT3 expression, and drastically attenuated the expression of proinflammatory mediators such as TNF-α, CCL2, and CXCL10 (Fig. 6B and C).Interestingly, additional inhibitory effects were noted for those treated with both amlexanox and SPI, suggesting a potential for novel drug combinations in the treatment of neuroinflammation-associated diseases.Based on these findings, we demonstrated that amlexanox reduces neuroinflammatory responses, at least partly depending on the inhibition of STAT3 signaling pathways.Despite a considerable understanding of amlexanox and the molecular markers examined in this work, further studies on either ectopic expression or pharmacological activation of IKKε or TBK1 are needed to fully elucidate the negative feedback, coregulation, and competitive binding mechanisms that govern their related signaling behaviors.
As the emergence of activated microglia is a common hallmark of neurodegenerative diseases, different approaches have explored ways to prevent or modulate microglial activation.Our results revealed that amlexanox (an aphthous ulcer drug) showed potent anti-inflammatory activity in LPS-induced microglia activation and neuroinflammatory murine model.This effect was mediated via complex pathways including NF-κB and STAT3 signaling pathways (Fig. 8).These findings provide novel insights into the anti-inflammatory mechanism of amlexanox and formulate the theoretical foundation for ameliorating neuroinflammation-related diseases, especially neurodegenerative diseases accompanied by activated microglia.Therefore, amlexanox might be considered a novel therapeutic strategy for neuroinflammatory and neurodegenerative diseases.

Animal care, ethics and treatments
Seven-week-old C57BL/6 male mice were purchased from Samtako (South Korea).Mice were maintained under standard conditions (24 ± 2 °C, 12 h day-night cycle, 50 ± 5% humidity) in a pathogen-free environment.Mice were allowed free access to food pellets and water throughout the study period.Experimental procedures and animal management procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Jeonbuk National University in Korea (approval number: NON2023-111).All methods were conducted following IACUC ethical guidelines and regulations supported by the Korean Council on Animal Care and Korean Animal Protection Law, 2007; Article 13 (Experiments with animals).All animal trials followed the ARRIVE 2.0 guidelines, including study design, animal numbers, randomization, and statistical methodologies.

Cell viability
Cell viability was assessed using Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan).BV2 cells were seeded in 96-well plates (100 μL per well, 5000 cells/well) for 24 h before experimental treatment.The cell was then incubated with increasing concentrations of amlexanox in the absence or presence of LPS (100 ng/mL).Following treatment, 10 µL of CCK-8 solution was added to each well and incubated for 2 h in the cell culture incubator.
Absorbance was measured at 450 nm using microtiter plate reader EMax spectrophotometry (Molecular Devices, Sunnyvale, CA).The total RNA was extracted from BV2 cells using GeneAll RNA extraction kit (GeneAll Biotechnology, Korea) and the purity of RNA was measured with NanoDrop 2000 (Thermol Scientific, USA).The RNA was reverse transcribed into cDNA using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Japan).qPCRBIO SyGreen Blue Mix (PCR Biosystems, London, UK) was used for real-time PCR on a CFX96™ Real-Time PCR System (BioRad, Hercules, CA).After the reaction was completed, the specificity of the amplified product was confirmed by a melting curve analysis.Gene expression quantification was performed by comparing the Ct value (cycle threshold value) of each sample normalized to the Ct value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Sequences of PCR primers are listed in Table 1.

Western blotting
To determine how amlexanox affects inflammatory signaling pathways, BV2 microglial cells were pre-treated with amlexanox or vehicle (< 0.1% DMSO) for 1 h, followed by 100 ng/mL LPS for 23 h.Thereafter, total protein was extracted from the BV2 cells using RIPA lysis buffer (iNtRON, Korea).Following centrifugation at 13,000 g for 20 min at 4 °C, the protein concentration in the supernatant was determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific Inc.), according to the manufacturer's instructions.Equal amounts of protein in each sample were then loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
After transferring protein from gel to PVDF membrane (Bio-Rad Laboratories), blocking was conducted using everyblot blocking buffer (Bio-Rad Laboratories).The membrane was then incubated overnight with primary antibodies at 4 °C.To detect the antigen-antibody complexes, HRP-conjugated secondary antibodies (Cell Signaling Technology) were diluted 1:1000, and incubated for 1 h at room temperature.All antibodies were diluted in everyblot blocking buffer.The protein bands were visualized using ImageQuant™ LAS 500 (GE Healthcare Life Science).Expression levels of protein were quantified with ImageJ software.

Enzyme-linked immunosorbent assay
IL-6, IL-1β, and TNF-α levels in serum and brain tissue lysates were determined using commercial ELISA kits (eBioscience, San Diego, CA, USA).All experiments were performed according to the manufacturer's instructions.

Immunohistochemistry (IHC)
For IHC staining, brains were fixed in 10% phosphate-buffered formalin, routinely processed, and then embedded in paraffin.Brain sections were 4 µm thick sliced and placed onto glass slides.Before the staining protocol, slides were deparaffinized, rehydrated, and submerged in antigen retrieval solution (Dako, Jena, Germany) for 30 min at 100 °C.Non-specific binding was blocked with 3% peroxidase solution followed by blocking with Super Block (ScyTek Laboratories, Inc., Logan, UT, USA).The slides were incubated with rabbit anti-Iba-1(A19776, ABclonal) antibodies at 4 °C overnight.And then, were further incubated with horseradish peroxidase-conjugated secondary antibody (Vector Laboratories, Newark, CA, USA).Immune complexes were detected using DAB Substrate Kit (Vector Laboratories) according to the manufacturer's instructions.Finally, tissues were counterstained and mounted.Images were analyzed under a light microscope (BX53F, Olympus Corp.) and digital imaging software (analySIS TS, Olympus Corp.).

Figure 2 .
Figure 2. Effects of amlexanox on TBK1/IKKε and proinflammatory enzymes (iNOS and COX-2) in LPSstimulated BV2 microglial cells.(A) Effects of amlexanox on the cell viability of BV2 microglia with or without LPS stimulation.Cells were then treated with increasing concentrations of amlexanox in the presence or absence of LPS (100 ng/mL) for 24 h, and viability was determined using the CCK-8 assay (n = 8).(B, C) Effects of amlexanox on TBK1 and IKKε in LPS-stimulated BV2 microglial cells.Following treatment with amlexanox for 1 h, the BV2 cells were stimulated with LPS for treatment for 6 h.The mRNA (B) and protein (C) expression levels of TBK1/p-TBK1 and IKKε in BV2 cells were determined by qRT-PCR and western blot (n = 4).(D) The mRNA and protein expression levels of iNOS and COX-2 in BV2 cells were determined by qRT-PCR (n = 4).(E) The protein expression levels of iNOS and COX-2 were determined by western blot (n = 4).(F) The mRNA expression levels of iNOS and COX-2 in BMDM were determined by qRT-PCR (n = 4).β-actin was used as an internal control for western blot analysis.The results are presented as the mean ± SD. #### p < 0.0001, compared with the control group; **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the LPS-treated control group.

Figure 3 .
Figure 3. Effects of amlexanox on proinflammatory mediators in LPS-induced murine BV2, BMDM, and HMC3 human microglial cells.The cell was pre-treated with amlexanox for 1 h, followed by treatment with LPS (100 ng/mL).After 6 h, qRT-PCR was performed to measure the mRNA expression of the proinflammatory mediators.(A) The mRNA expression levels of pro-inflammatory mediators in LPS-induced BV2 microglia.(B) The mRNA expression levels of pro-inflammatory mediators in LPS-induced BMDM.(C) The mRNA expression levels of pro-inflammatory mediators in LPS-induced HMC3.GAPDH was used as an internal control for real-time PCR.Data are shown as the mean ± SD (n = 4).#### p < 0.0001, compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the LPS-treated control group.

Figure 4 .
Figure 4. Effects of amlexanox on AKT and MAPKs in LPS-stimulated BV2 microglial cells.Cells were pretreated with amlexanox for 1 h, followed by stimulation with LPS for 23 h.Whole-cell extracts were prepared, and analyzed by immunoblotting using AKT or MAPKs antibodies and their phosphorylated forms.The results are presented as the mean ± SD (n = 3).## p < 0.01, and #### p < 0.0001, compared with the control group; *p < 0.05, and **p < 0.01, compared with the LPS-treated control group.

Figure 6 .
Figure 6.Effects of amlexanox on STAT3 signaling pathways in LPS-stimulated BV2 microglial cells.(A) BV2 cells were treated with various concentrations of amlexanox for 1 h before treatment with LPS (100 ng/mL) for 23 h for western blotting (n = 3).(Band C) BV2 microglial cells were treated with SPI (STAT3 inhibitor, 5 μM) for 1 h, then with amlexanox (6 μM) for 1 h, and finally treated with LPS (100 ng/mL) and incubated for the indicated time.Real-time PCR and western blot were performed to measure the expression level of STAT3 and proinflammatory mediators (n = 3).GAPDH was used as an internal control for real-time PCR.Data are shown as the mean ± SD. ## p < 0.01, ### p < 0.001, and #### p < 0.0001, compared with the control group; *p < 0.05, compared with the LPS-treated control group.